JPH0530985A - Production of l-lysine by fermentation - Google Patents

Abstract

PURPOSE:To produce L-lysine in improved productivity and accumulation concen tration in a high yield by inoculating a L-lysine-producing strain on a liquid medium, culturing the strain, feeding a feed medium at a specific time so as to maintain the concentration of carbon at a specified value, and simultaneously culturing the strain. CONSTITUTION:A lysine-producing microorganism (e.g. Brevibacterium.lactofermentum ATCC 21800) is inoculated on a perfect liquid medium, cultured and subjected to a logarithmic growth. The culture solution is subsequently mixed with a feed medium containing both a carbon source (e.g. sugar or organic acid) and a nutrient having a growth-accelerating effect (e.g. soy bean protein hydrolysate or yeast extract) and is fed to culture so that the concentration of the carbon source in the culture solution is maintained at <=5g/l, followed by collecting the objective L-lysine.

Description

Detailed Description of the Invention

BACKGROUND OF THE INVENTION The present invention relates to the production of L-lysine, which is an important amino acid used as an additive in feed for broilers and pigs by fermentation, due to lack in feed grains such as corn. It is about law.

2. Description of the Prior Art L- by a conventionally known fermentation method
The lysine production method comprises culturing an organism having L-lysine-producing ability in a batch system or a continuous system, accumulating L-lysine in a medium, and collecting this. In case of batch method,
A liquid medium containing a carbon source or a nitrogen source is put into a fermenter for batch culture, or a medium containing only a carbon source is continuously or read-added for fed-batch culture. In the case of the continuous system, the culture medium is continuously supplied to the fermentation tank, the same amount of the culture solution is continuously taken out, and the culture is carried out while maintaining a constant steady value such as the bacterial cell amount and the product concentration in the tank. ..

When performing L-lysine fermentation, a conventional batch system can obtain high values for the concentration and yield of the product in the culture broth, but high productivity. It is difficult to obtain a value, while in the conventional continuous method, it is possible to obtain a high value for productivity, but it is difficult to obtain a high value for the concentration and yield of the product. However, in order to meet the increasing demand for L-lysine and to produce it at a lower cost, it is necessary to increase the productivity of L-lysine fermentation more than ever, and to increase the product accumulation concentration and yield. Under such a technical background, the object of the present invention is to achieve high productivity, accumulated product concentration, and yield by a novel fermentation method that combines the advantages of the conventional batch system and continuous system. This is to provide a method for producing L-lysine.

Means for Solving the Problems As a result of various studies on the method of L-lysine fermentation by L-lysine-producing bacteria, the present inventor has changed L-lysine-producing bacteria to a liquid medium containing a carbon source and a nitrogen source. Inoculate and continue culturing while feeding a feed medium containing both a carbon source and nutrients having a growth promoting effect after the end of logarithmic growth so that the concentration of the carbon source in the culture solution is maintained at 5 g / L or less. As a result, while maintaining the same high product accumulation concentration and high yield as in the conventional batch fermentation, L- which has high productivity in continuous reading fermentation.
It was discovered that lysine fermentation was possible, and the present invention was completed based on this finding. That is, the present invention inoculates a liquid medium with a microorganism having an L-lysine-producing ability, and a feed medium containing both a carbon source and a nutrient having a growth-promoting effect in the culture medium after the logarithmic growth of the microorganism is finished. The present invention relates to a method for producing L-lysine by a fermentation method, which comprises culturing while feeding so that the concentration of a carbon source is maintained at 5 g / L or less, and collecting L-lysine produced and accumulated in a culture solution. Hereinafter, the present invention will be sequentially described. In the method of the present invention, the microorganism capable of producing L-lysine is not particularly limited, and any microorganism can be used as long as it is a characteristic organism capable of producing L-lysine. Such microorganisms include, for example, properties necessary for imparting L-lysine productivity (homoserine requirement, S- (2-aminoethyl) -L
-Cysteine resistance, α-chlorocaprolactam resistance, etc.)
A microorganism belonging to the genus Brevibacterium or the genus Corynebacterium having, more specifically, for example, Brevibacterium lactofermentum ATCC 21800, Brevibacterium flavum ATCC 21475, and Corynebacterium acetoglutamicum ATCC 21491 can be mentioned. The liquid medium is also not particularly limited, and a conventionally known complete liquid medium containing organic and inorganic nutrients such as carbon source and nitrogen source and other micronutrients can be used. The carbon source for feed used in the method of the present invention may be one commonly used as a fermentation raw material (carbon source) such as sugar, organic acid and alcohol, as long as it is a carbon source that can be assimilated by the lysine-producing bacterium. Anything is fine. Further, the nutrient having a growth promoting effect refers to amino acids, vitamins and natural products containing them which are effective in accelerating the growth of L-lysine-producing bacteria. Specific examples are soy protein hydrolyzate, yeast extract, corn steep liquor and the like. Now, as described above, the medium fed in the conventional technique is a medium which usually contains only a carbon source in the fed-batch culture, and is a complete liquid medium in the continuous system. On the other hand, the medium to be fed in the method of the present invention is a medium containing both a carbon source and nutrients having a growth promoting effect, and such a feed medium is one of the features of the method of the present invention. Is. In the method of the present invention, the medium to be fed is fed after the end of the logarithmic growth of the microorganism. Feeding before the end of logarithmic growth may impair the initial growth of the bacterium and should be avoided.
The medium feeding method after the end of logarithmic growth may be continuous or intermittent. Further, when it is expected that the amount of the culture solution in the fermenter exceeds the allowable amount of the charged capacity of the fermenter by feeding, a part of the culture solution is preliminarily or when the allowable amount is reached. Further feeding can be performed by taking it out of the fermenter. So the important thing about media feed is
It is necessary to feed so that the carbon source concentration in the culture solution is always maintained at 5 g / L or less, which is also a feature of the method of the present invention. For this purpose, it is possible to employ a method of directly analyzing the carbon source concentration by sampling the culture solution at appropriate times, measuring the pH and dissolved oxygen concentration, and sensing the carbon source deficiency state from the changes to control the medium feed. It is also possible to adopt a method of doing so. If the carbon source concentration is not always maintained at 5 g / L or less, the growth of bacteria and the production rate of L-lysine will be reduced, and the object of the present invention will not be met. The features of the method of the present invention are as described above, and other fermentation conditions are not particularly limited. For example, the temperature for L-lysine fermentation by the method of the present invention may be any temperature at which the lysine-producing bacterium used can grow, and is usually 25 to 45 ° C, preferably 30 to 40 ° C, and the fermentation pH is , Usually 5.8-8.
5, preferably 6.5 to 7.5. Inorganic or organic acidic or alkaline substances, as well as urea, calcium carbonate, ammonia gas and the like can be used to adjust the pH. Further, the shape of the fermenter used in the present invention may be any shape as long as it is usually used for amino acid fermentation and the like, a complete mixing tank equipped with turbine blades,
An air lift type fermenter can be used. Further, L-lysine can be collected from the fermented liquid after the culture by a conventionally known method such as an ion exchange resin method or a crystallization method, or a combination thereof.

EXAMPLES The present invention will be further described below with reference to examples. Example 1 Glucose 30 g / L, ammonium chloride 10 g / L,
Urea _{3g / L, KH 2 PO 4} 1g / L, MgSO 4 · 7
_{H 2 O100mg / L, FeSO 4} · 7H 2 O10mg
/ L, MnSO ₄ .4H ₂ O 8 mg / L, soybean protein hydrolyzate (as nitrogen) 1 g / L, thiamine hydrochloride 0.1 mg / L and biotin 0.3 mg / L (pH 7.0). , 500 mL shake flask 3
20 mL each was dispensed into the book. After heat sterilization at 115 ° C for 10 minutes, 1 platinum loop of Brevibacterium lactofermentum ATCC 21800 which had been grown on broth slant for 48 hours was inoculated into this medium at 31.5 ° C.
The cells were shake-cultured for 24 hours. The above is the seed culture. 80 g / L of waste sugar concentrate as sugar, 50 g of ammonium sulfate /
_{L, KH 2 PO 4 1g /} L, MgSO 4 · 7H 2 O1g
/ L, soy protein hydrolyzate (as nitrogen) 100mg
/ L, 0.1 mg / L of thiamine hydrochloride and 0.3 mg / L of biotin (pH 7.0) were added to three 1 L glass small fermenters in 300 mL aliquots,
It heat-sterilized at 120 degreeC for 15 minutes. After cooling to 31.5 ° C, the above flask culture completed liquid is added to the fermenter at 15 m
L at a temperature of 31.5 ° C, aeration rate of 1/2 vvm,
Culturing was performed under the condition of stirring at 700 rpm. For one of the three, the culture was terminated when the sugar in the culture medium was exhausted (conventional batch culture), and the concentration of L-lysine accumulated in the culture medium was measured by the acid-copper ninhydrin coloring method. Was quantified by For the other two groups, the sugar concentration in the culture was 5
Feeding of the feed medium was started at the time when the amount became g / L or less. For one of these, the feed medium contains only glucose (40 g / dL) (conventional fed-batch culture), for the other one, the medium contains glucose (40 g / dL).
/ DL), soy protein hydrolyzate (100m as nitrogen
g / L), thiamine hydrochloride (0.1 mg / L) and biotin (0.3 mg / L) (invention).
In both cases, the culture can be read while adjusting the feed rate of the feed medium so that the sugar concentration in the culture solution is 5 g / L or less,
After feeding 100 mL of each feed medium,
When the sugar in the culture solution is consumed, the culture is finished,
The concentration of L-lysine accumulated in the culture solution was quantified. The results of three cultures are shown in Table 1.

[Table 1] It can be seen from Table 1 that the method of the present invention is excellent in both productivity and yield of L-lysine. Example 2 Glucose 30 g / L, ammonium chloride 10 g / L,
Urea _{3g / L, KH 2 PO 4} 1g / L, MgSO 4 · 7
_{H 2 O100mg / L, FeSO 4} · 7H 2 O10mg
/ L, MnSO ₄ .4H ₂ O 8 mg / L, soy protein hydrolyzate (as nitrogen) 1 g / L, thiamine hydrochloride 0.1 mg / L, and biotin 0.3 mg / L (pH 7.0) Was dispensed into two 500 mL shake flasks, 20 mL each. After heat sterilization at 115 ° C for 10 minutes, this medium was preliminarily sown on a broth slant for 48 minutes.
Brevibacterium flavum ATCC grown for hours
One platinum loop was inoculated with 21475 and cultured at 31.5 ° C. for 24 hours with shaking. The above is the seed culture. 80 g / L as waste sugar concentrate, 50 g / L ammonium sulfate, KH _{2
PO 4 1g / L, MgSO 4} · 7H 2 O1g / L, soybean protein hydrolyzate (as nitrogen) 100 mg / L, thiamine hydrochloride 0.1 mg / L, and biotin 0.3mg
A medium (pH 7.0) containing / L was dispensed into two 1-L small glass fermenters each in an amount of 300 mL and sterilized by heating at 120 ° C for 15 minutes. After cooling to 31.5 ° C., 15 mL of the above flask culture completed liquid was added to each fermenter, and the culture was carried out under the conditions of a temperature of 31.5 ° C., an aeration rate of 1/2 vvm, and a stirring number of 700 rpm. .. Feeding of the feed medium was started when the sugar concentration in the culture solution became 5 g / L or less. The feed medium is glucose (40 g / d
L), soy protein hydrolyzate (100 mg / nitrogen /
L), thiamine hydrochloride (0.1 mg / L) and biotin (0.3 mg / L). One of the fermenters continued to cultivate while adjusting the feed rate of the feed medium so that the sugar concentration in the medium was always 5 g / L or less (the present invention), and the other had a sugar concentration of 5 to 15 g / L. Culture was continued at the same feed rate (control), 100m each
After the feed culture of L was completed, the culture was terminated when the sugar in the culture medium was consumed, and the concentration of L-lysine accumulated in the culture medium was quantified. The results of culturing in two fermenters are shown in Table 2.

[Table 2] Example 3 Glucose 30 g / L, ammonium chloride 10 g / L,
Urea _{3g / L, KH 2 PO 4} 1g / L, MgSO 4 · 7
_{H 2 O100mg / L, FeSO 4} · 7H 2 O10mg
/ L, MnSO ₄ .4H ₂ O 8 mg / L, soy protein hydrolyzate (as nitrogen) 1 g / L, thiamine hydrochloride 0.1 mg / L, and biotin 0.3 mg / L (pH 7.0) 20 mL was dispensed into a 500 mL shake flask. After heat sterilization at 115 ° C for 10 minutes,
Brevibacterium flavum ATCC 2147 was grown in this medium on broth slants for 48 hours.
One platinum loop was inoculated with 5 and shake-cultured at 31.5 ° C. for 24 hours. The above is the seed culture. 80g of waste sugar concentrate as sugar
/ L, ammonium sulfate 50g / L, KH ₂ PO ₄ 1g
_{/ L, MgSO 4 · 7H 2} O1g / L, soybean protein hydrolyzate (nitrogen as) 100 mg / L, thiamine hydrochloride 0.1 mg / L, and medium containing biotin 0.3 mg / L (pH 7.0) 300 mL was dispensed into a 1 L glass small fermentor and sterilized by heating at 120 ° C. for 15 minutes. After cooling to 31.5 ° C., 15 mL of the above flask culture completed solution (seed culture solution) was added to the fermenter, and the temperature was adjusted to 31.
The culture was carried out under the conditions of 5 ° C., an aeration rate of 1/2 vvm, and a stirring number of 700 rpm. Feeding of the feed medium was started when the sugar concentration in the culture solution became 5 g / L or less. The feed medium was glucose (40 g / dL), soy protein hydrolyzate (100 mg / L as nitrogen), thiamine hydrochloride (0.1 mg / L) and biotin (0.3 mg / L).
L) was contained. The sugar concentration in the culture solution is always 5 g / L
The culture was continued while adjusting the feed rate of the feed medium as follows, and when 100 mL of the feed medium had been fed, when the sugar in the culture solution was consumed, the culture solution 1
00 mL was withdrawn from the culture tank. Further, the culture was continued while feeding the feed medium. Also in this case, the sugar concentration in the culture solution was adjusted to 5 g / L or less. After 100 mL of the feed medium had been fed, the culture was terminated when the sugar in the culture solution was exhausted, and the culture solution in the fermenter was combined with the previously extracted culture solution to collect L accumulated therein. -The lysine concentration was quantified. As a result, the productivity of lysine was 3.8 g /
L · hr, and the lysine yield was 42%. Example 4 Glucose 30 g / L, ammonium chloride 10 g / L,
Urea _{3g / L, KH 2 PO 4} 1g / L, MgSO 4 · 7
_{H 2 O100mg / L, FeSO 4} · 7H 2 O10mg
/ L, MnSO ₄ .4H ₂ O 8 mg / L, soy protein hydrolyzate (as nitrogen) 1 g / L, thiamine hydrochloride 0.1 mg / L, and biotin 0.3 mg / L (pH 7.0) Was dispensed into two 500 mL shake flasks, 20 mL each. After heat sterilization at 115 ° C for 10 minutes, this medium was preliminarily sown on a broth slant for 48 minutes.
One platinum loop was inoculated with Corynebacterium acetoacidophilum ATCC 21491 grown for 3 hours, and then 31.5
The cells were shake-cultured at 24 ° C for 24 hours. The above is the seed culture.
80 g / L of waste sugar concentrate as sugar, 50 g of ammonium sulfate
_{/ L, KH 2 PO 4 1g} / L, MgSO 4 · 7H 2 O1
g / L, soy protein hydrolyzate (as nitrogen) 100m
g / L, 0.1 mg / L of thiamine hydrochloride, and 0.3 mg / L of biotin (pH 7.0) were added to 2 media.
300 mL each was dispensed to the base 1 L small glass fermentor and heat-sterilized at 120 ° C. for 15 minutes. After cooling to 31.5 ° C., the flask culture completed liquid was added to each fermenter for 15 minutes.
Add each mL, temperature 31.5 ℃, aeration rate 1/2 vv
The culture was performed under the conditions of m. In one of the fermenters, the feed of the feed medium was started when the sugar concentration in the culture solution became 5 g / L or less. The feed medium contained glucose (40 g / dL), soy protein hydrolyzate (100 mg / L as nitrogen), thiamine hydrochloride (0.1 mg / L) and biotin (0.3 mg / L). The culture is continued while adjusting the feed rate of the feed medium so that the sugar concentration in the culture solution is always 5 g / L or less, and when 100 mL of the feed medium is completely fed, the sugar in the culture solution is consumed. Culture medium 100m
L was removed from the culture tank. Further, the culture was continued while feeding the feed medium. Also in this case, the sugar concentration in the culture solution was adjusted to 5 g / L or less. After 100 mL of the feed medium had been fed, the culture was terminated when the sugar in the culture solution was exhausted, and the culture solution in the fermenter was combined with the previously extracted culture solution to collect L accumulated therein. -The lysine concentration was quantified. As a result, the productivity of lysine was 3.9 g / L · h.
and the lysine yield was 41%. In the other fermentor, the addition of the continuous culture fed-batch medium was started 25 hours after the start of culture. As the feeding medium, a 4-fold diluted starting medium was used. About 1 L of a fed-batch medium was flown at a dilution rate of 0.05 hr ⁻¹ to achieve a steady state by continuous culture, and then the L-lysine concentration in the culture solution was quantified.
As a result, the lysine productivity was 3.5 g / L · hr, and the lysine yield was 35%.

Industrial Applicability According to the present invention, L-lysine fermentation can be carried out while maintaining the high product accumulation concentration and high yield as in the conventional batch system fermentation and having the high productivity as in the conventional continuous system. Became. In other words, it has become possible to significantly improve the industrial production of L-lysine and reduce the cost.

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[Procedure amendment]

[Submission date] December 19, 1991

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Detailed explanation of the invention

[Correction method] Change

[Correction content]

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to the production of L-lysine, which is an important amino acid used as an additive in feed for broilers and pigs by fermentation, due to lack in feed grains such as corn. It is about law.

[0002]

2. Description of the Prior Art L- by a conventionally known fermentation method
The method for producing lysine is to culture a microorganism capable of producing L-lysine in a batch system or a continuous system to accumulate L-lysine in a medium and collect the L-lysine.

In the case of the batch system, a liquid medium containing a carbon source or a nitrogen source is put into a fermenter for batch culture, or a medium containing only a carbon source is continuously or intermittently added and fed. Incubate. In the case of the continuous system, the culture medium is continuously supplied to the fermentation tank, the same amount of the culture solution is continuously taken out, and the culture is carried out while maintaining a constant steady value such as the bacterial cell amount and the product concentration in the tank. ..

[0004]

When performing L-lysine fermentation, a conventional batch system can obtain high values for the concentration and yield of the product in the culture broth, but high productivity. It is difficult to obtain a value, while in the conventional continuous method, it is possible to obtain a high value for productivity, but it is difficult to obtain a high value for the concentration and yield of the product. However, in order to meet the increasing demand for L-lysine and to produce it at a lower cost, it is necessary to increase the productivity of L-lysine fermentation more than ever, and to increase the product accumulation concentration and yield.

Under such a technical background, the object of the present invention is to improve productivity, accumulated product concentration and yield by the novel fermentation method which has the advantages of both the conventional batch system and continuous system. It is to provide a method for producing L-lysine which is expensive.

[0006]

Means for Solving the Problems The present inventor has conducted various researches on a method of L-lysine fermentation by an L-lysine-producing bacterium, and as a result, the L-lysine-producing bacterium is a liquid medium containing a carbon source and a nitrogen source. And a feed medium containing both a carbon source and a nutrient having a growth promoting effect after the end of logarithmic growth is fed while maintaining the concentration of the carbon source in the culture solution at 5 g / L or less. By continuing, while maintaining the same high product accumulation concentration and high yield as in conventional batch fermentation, L- with high productivity in continuous fermentation
It was discovered that lysine fermentation was possible, and the present invention was completed based on this finding.

That is, the present invention inoculates a liquid medium with a microorganism capable of producing L-lysine, and cultures a feed medium containing both a carbon source and nutrients having a growth promoting effect after the end of logarithmic growth of the microorganism. The concentration of carbon source in the liquid is 5
The present invention relates to a method for producing L-lysine by a fermentation method, which comprises culturing while feeding so as to be maintained at g / L or less and collecting L-lysine produced and accumulated in the culture solution.

The present invention will be sequentially described below.

In the method of the present invention, there is no particular limitation on the microorganism capable of producing L-lysine, and any microorganism can be used as long as it is capable of producing L-lysine. Examples of such a microorganism include, for example, properties necessary for imparting L-lysine productivity (homoserine requirement, S-
(2-aminoethyl) -L-cysteine resistance, α-chlorocaprolactam resistance, etc.) can be mentioned as a microorganism belonging to the genus Brevibacterium or the genus Corynebacterium. More specifically, for example, Brevibacterium lactofermentum ATCC 2180
0, Brevibacterium flavum ATCC 2147
5, and Corynebacterium acetoglutamicum AT
CC 21491 can be mentioned.

The liquid medium is not particularly limited, and a conventionally known complete liquid medium containing organic and inorganic nutrients such as carbon source and nitrogen source and other micronutrients can be used.

The carbon source for feed used in the method of the present invention is generally used as a fermentation raw material (carbon source) such as sugar, organic acid and alcohol, as long as it is a carbon source that can be assimilated by the lysine-producing bacterium. Anything will do. Also,
The nutrient having a growth promoting effect refers to amino acids, vitamins and natural products containing them which are effective in accelerating the growth of L-lysine-producing bacteria. Specific examples are soy protein hydrolyzate, yeast extract, corn steep liquor and the like.

The medium fed in the conventional technique is a medium which normally contains only a carbon source in the fed-batch culture as described above, and is a complete liquid medium in the continuous system. On the other hand, the medium to be fed in the method of the present invention is a medium containing both a carbon source and nutrients having a growth promoting effect, and such a feed medium is one of the features of the method of the present invention. Is.

In the method of the present invention, the medium to be fed is fed after the end of the logarithmic growth of the microorganism. Feeding before the end of logarithmic growth may impair the initial growth of the bacterium and should be avoided.

The method of feeding the medium fed after the end of logarithmic growth may be continuous or intermittent. Further, when it is expected that the amount of the culture solution in the fermenter exceeds the allowable amount of the charged capacity of the fermenter by feeding, a part of the culture solution is preliminarily or when the allowable amount is reached. Further feeding can be performed by taking it out of the fermenter.

However, what is important regarding the feeding of the medium is that it is necessary to feed so that the carbon source concentration in the culture solution is always kept at 5 g / L or less, which is also a feature of the method of the present invention. Is. For this purpose, it is possible to employ a method of directly analyzing the carbon source concentration by sampling the culture solution at appropriate times, measuring the pH and dissolved oxygen concentration, and sensing the carbon source deficiency state from the changes to control the medium feed. It is also possible to adopt a method of doing so. If the carbon source concentration is not always kept below 5 g / L, the growth of bacteria and the production rate of L-lysine decrease,
After all, the purpose of the present invention will not be met.

The characteristics of the method of the present invention are as described above, and other fermentation conditions and the like are not particularly limited. For example, the temperature for L-lysine fermentation by the method of the present invention may be any temperature at which the lysine-producing bacterium used can grow, and is usually 25 to 45 ° C, preferably 30 to 40 ° C, and the fermentation pH is , Usually 5.8 to 8.5, preferably 6.
It is 5 to 7.5. To adjust the pH, inorganic or organic acidic or alkaline substances, as well as urea, calcium carbonate,
Ammonia gas or the like can be used. The shape of the fermenter used in the present invention may be any shape as long as it is usually used for amino acid fermentation and the like,
A complete mixing tank equipped with turbine blades, an air lift type fermentation tank, etc. can be used.

L-lysine can also be collected from the fermented liquid after the culture by a conventionally known method such as an ion exchange resin method or a crystallization method, or a combination thereof.

[0018]

EXAMPLES The present invention will be further described below with reference to examples. Example 1 Glucose 30 g / L, ammonium chloride 10 g / L,
Urea _{3g / L, KH 2 PO 4} 1g / L, MgSO 4 · 7
_{H 2 O100mg / L, FeSO 4} · 7H 2 O10mg
/ L, MnSO ₄ . 4H ₂ O 8mg / L, soybean protein hydrolyzate (as nitrogen) 1g / L, thiamine hydrochloride 0.1mg / L and biotin 0.3mg / L containing medium (pH 7.0) 500mL shake flask Three
20 mL each was dispensed into the book. After heat sterilization at 115 ° C for 10 minutes, 1 platinum loop of Brevibacterium lactofermentum ATCC 21800 which had been grown on broth slant for 48 hours was inoculated into this medium at 31.5 ° C.
The cells were shake-cultured for 24 hours. The above is the seed culture.

[0019] The waste molasses as a sugar 80g / L, ammonium sulfate _{50g / L, KH 2 PO 4} 1g / L, MgSO 4
・ 7H ₂ O ₁ g / L, soybean protein hydrolyzate (as nitrogen) 100 mg / L, thiamine hydrochloride 0.1 mg / L
And a medium containing 0.3 mg / L of biotin (pH 7.
0) 300 mL in 3 small 1 L glass fermenters
Each was dispensed and heat sterilized at 120 ° C. for 15 minutes. 31.
After cooling to 5 ° C., the above-mentioned flask culture completed liquid was added to fermenter
Add 15mL per group, temperature 31.5 ℃, aeration 1
The culture was carried out under the condition of / 2 vvm and the stirring number was 700 rpm.

For one of the three, the culture was terminated when the sugar in the culture solution was exhausted (conventional batch culture), and the concentration of L-lysine accumulated in the culture solution was adjusted to acidic-copper. It was quantified by the ninhydrin color development method. For the other two groups, the feeding of the feed medium was started when the sugar concentration in the culture solution became 5 g / L or less. For one of these, the feed medium contains only glucose (40 g / dL) (conventional fed-batch culture), for the other one,
The medium contained glucose (40 g / dL), soybean protein hydrolyzate (100 mg / L as nitrogen), thiamine hydrochloride (0.1 mg / L) and biotin (0.3 mg / L) (invention). .. In both cases, the culture was continued while adjusting the feed rate of the feed medium so that the sugar concentration in the culture solution was 5 g / L or less, and after the completion of feeding 100 mL of each feed medium, the sugar in the culture solution was consumed up. At that point, the culture was terminated and the concentration of L-lysine accumulated in the culture solution was quantified.

The results of three cultures are shown in Table 1.

[0022]

[Table 1] It can be seen from Table 1 that the method of the present invention is excellent in both productivity and yield of L-lysine. Example 2 Glucose 30 g / L, ammonium chloride 10 g / L,
Urea _{3g / L, KH 2 PO 4} L, MgSO 4 · 7H 2 O
_{100mg / L, FeSO 4 · 7H} 2 O10mg / L,
MnSO ₄ .4H ₂ O 8 mg / L, soybean protein hydrolyzate (as nitrogen) 1 g / L, thiamine hydrochloride 0.1 m
A medium (pH 7.0) containing g / L and 0.3 mg / L of biotin was dispensed into two 500 mL shake flasks, 20 mL each. After heat sterilization at 115 ° C. for 10 minutes, Brevibacterium flavum ATCC 21 preliminarily grown on broth slant for 48 hours was added to this medium.
One platinum loop was inoculated with 475 and cultured at 31.5 ° C. for 24 hours with shaking. The above is the seed culture.

[0023] 80g / L of waste molasses as sugar, ammonium sulfate _{50g / L, KH 2 PO 4} 1g / L, MgS
O ₄ . 7H ₂ O ₁ g / L, soy protein hydrolyzate (as nitrogen) 100 mg / L, thiamin hydrochloride 0.1 mg
/ L and medium containing 0.3 mg / L of biotin (p
H7.0) into two 1L glass small fermenters
It was dispensed in 0 mL aliquots and sterilized by heating at 120 ° C for 15 minutes.
After cooling to 31.5 ° C, 15 mL of the above flask culture completed liquid was added to each fermentor, and the temperature was 31.5 ° C.
The culture was performed under the conditions of an aeration rate of 1/2 vvm and a stirring rate of 700 rpm.

Feeding of the feed medium was started when the sugar concentration in the culture solution became 5 g / L or less. The feed medium contained glucose (40 g / dL), soy protein hydrolyzate (100 mg / L as nitrogen), thiamine hydrochloride (0.1 mg / L) and biotin (0.3 mg / L). One fermentor always had a sugar concentration of 5 in the medium.
Culturing is continued while adjusting the feed rate of the feed medium so as to be g / L or less (the present invention), and the other is 5 to 15
Cultivation was continued at a feed rate such that the sugar concentration was g / L (control), and after each 100 mL of feed culture had been fed, the culture was terminated when the sugar in the culture medium was consumed, and The L-lysine concentration accumulated therein was quantified.

The results of culturing in two fermenters are shown in Table 2.

[0026]

[Table 2] Example 3 Glucose 30 g / L, ammonium chloride 10 g / L,
Urea _{3g / L, KH 2 PO 4} 1g / L, MgSO 4 · 7
_{H 2 O100mg / L, FeSO 4} · 7H 2 O10mg
/ L, MnSO ₄ . 500 mL of a medium (pH 7.0) containing 4 mg / L of 4H ₂ O, 1 g / L of soybean protein hydrolyzate (as nitrogen), 0.1 mg / L of thiamine hydrochloride, and 0.3 mg / L of biotin was shaken. 20 mL was dispensed into the flask. After heat sterilization at 115 ° C for 10 minutes,
Brevibacterium flavum ATCC 2147 was grown in this medium on broth slants for 48 hours.
One platinum loop was inoculated with 5 and shake-cultured at 31.5 ° C. for 24 hours. The above is the seed culture.

[0027] The waste molasses as a sugar 80g / L, ammonium sulfate _{50g / L, KH 2 PO 4} 1g / L, MgSO 4
7H ₂ O ₁ g / L, soybean protein hydrolyzate (as nitrogen) 100 mg / L, thiamin hydrochloride 0.1 mg /
L, and medium containing 0.3 mg / L of biotin (pH
7.0) was dispensed into a 1 L glass small fermentor in an amount of 300 mL, and sterilized by heating at 120 ° C. for 15 minutes. After cooling to 31.5 ° C., 15 mL of the above flask culture completed liquid (seed culture liquid) was added to the fermentor, and the temperature was 31.5 ° C.
The culture was carried out under the conditions of 2 vvm and a stirring number of 700 rpm.

Feeding of the feed medium was started when the sugar concentration in the culture solution became 5 g / L or less. The feed medium contained glucose (40 g / dL), soy protein hydrolyzate (100 mg / L as nitrogen), thiamine hydrochloride (0.1 mg / L) and biotin (0.3 mg / L). The culture is continued while adjusting the feed rate of the feed medium so that the sugar concentration in the culture solution is always 5 g / L or less, and when 100 mL of the feed medium is completely fed, the sugar in the culture solution is consumed. Culture medium 100m
L was removed from the culture tank.

Further, the culture was continued while feeding the feed medium. Also in this case, the sugar concentration in the culture solution was adjusted to 5 g / L or less. After 100 mL of the feed medium had been fed, the culture was terminated when the sugar in the culture solution was exhausted, and the culture solution in the fermenter was combined with the previously extracted culture solution to collect L accumulated therein. -The lysine concentration was quantified.

As a result, the productivity of lysine was 3.8 g / L.
-Hr and the lysine yield was 42%. Example 4 Glucose 30 g / L, ammonium chloride 10 g / L,
Urea _{3g / L, KH 2 PO 4} 1g / L, MgSO 4 · 7
_{H 2 O100mg / L, FeSO 4} · 7H 2 O10mg
/ L, MnSO ₄ .4H ₂ O 8 mg / L, soy protein hydrolyzate (as nitrogen) 1 g / L, thiamine hydrochloride 0.1 mg / L, and biotin 0.3 mg / L (pH 7.0) Was dispensed into two 500 mL shake flasks, 20 mL each. After heat sterilization at 115 ° C for 10 minutes, this medium was preliminarily sown on a broth slant for 48 minutes.
Corynebacterium aceto Gurutamiku <br/> beam ATCC twenty-one thousand four hundred and ninety-one obtained by time grow 1 loopful inoculated, 31.5 ° C.
The cells were shake-cultured for 24 hours. The above is the seed culture.

[0031] The waste molasses as a sugar 80g / L, ammonium sulfate _{50g / L, KH 2 PO 4} 1g / L, MgSO 4
7H ₂ O ₁ g / L, soybean protein hydrolyzate (as nitrogen) 100 mg / L, thiamin hydrochloride 0.1 mg /
L, and medium containing 0.3 mg / L of biotin (pH
7.0) in 2 small 1L glass fermenters 300
It was dispensed by mL and heat sterilized at 120 ° C. for 15 minutes. Three
After cooling to 1.5 ° C, 15 mL of the above flask culture completed liquid was added to each fermentor, and the culture was performed under the conditions of a temperature of 31.5 ° C, an aeration rate of 1/2 vvm, and a stirring number of 700 rpm.

In one fermenter, the sugar concentration in the culture solution was 5 g
Feeding of the feed medium was started from the time when it became less than / L. The feed medium is glucose (40 g / dL),
Soy protein hydrolyzate (100 mg / L as nitrogen),
It contained thiamine hydrochloride (0.1 mg / L) and biotin (0.3 mg / L). The culture is continued while adjusting the feed rate of the feed medium so that the sugar concentration in the culture solution is always 5 g / L or less, and when 100 mL of the feed medium is completely fed, the sugar in the culture solution is consumed. 100 mL of the culture solution was withdrawn from the culture tank.

Further, the culture was continued while feeding the feed medium. Also in this case, the sugar concentration in the culture solution was adjusted to 5 g / L or less. After 100 mL of the feed medium had been fed, the culture was terminated when the sugar in the culture solution was exhausted, and the culture solution in the fermenter was combined with the previously extracted culture solution to collect L accumulated therein. -The lysine concentration was quantified.

As a result, the productivity of lysine was 3.9 g / L.
-Hr and the lysine yield was 41%.

In the other fermentor, the continuous culture fed-batch medium was added 25 hours after the start of culture. As the feeding medium, a 4-fold diluted starting medium was used. About 1 L of a fed-batch medium was flown at a dilution rate of 0.05 hr ⁻¹ to achieve a steady state by continuous culture, and then the L-lysine concentration in the culture solution was quantified.

As a result, the productivity of lysine was 3.5 g / L.
-Hr and the lysine yield was 35%.

[0037]

Industrial Applicability According to the present invention, L-lysine fermentation can be carried out while maintaining the high product accumulation concentration and high yield as in the conventional batch system fermentation and having the high productivity as in the conventional continuous system. Became. In other words, it has become possible to significantly improve the industrial production of L-lysine and reduce the cost.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yosuke Yoyama 450 Morotomi, Morotomi-cho, Saga-gun, Saga Prefecture Inside the Ajinomoto Co., Inc. Kyushu Factory (72) Inventor Osamu Tosaka 450 Morotomizu, Morotomi-cho, Saga-gun, Saga Prefecture Nomoto Co., Ltd. Kyushu factory

Claims (1)

Claims: 1. A feed medium inoculating a liquid medium with a microorganism capable of producing L-lysine and containing both a carbon source and a nutrient having a growth promoting effect after the end of logarithmic growth of the microorganism. L-lysine by a fermentation method, in which L-lysine produced and accumulated in the culture solution is collected by culturing while feeding so that the concentration of the carbon source in the culture solution is maintained at 5 g / L or less. Manufacturing method.